31 Cell Cloning
313
Procedure
1. Select a culture containing cells to be cloned, and harvest the cells by Preparation
treating cells with an appropriate enzyme (see chapter 4, subprotocol of cells
1-3).
2. Wash the harvested cells with culture medium by centrifugation at
150 g for 5 min.
3. Suspend the cell pellet in culture medium.
1. Pour the cloning medium into a petri dish to a depth of 3-5 mm.
Dilution of cell
2. Add the cell suspension to the petri dish so as to make a cell density of suspension
10 cells/ml or 2 cells/cm 2 •
3. Culture the cells at the same temperature as that used for culturing
the cells.
1. When the cells have multiplied and formed colonies, select a colony. Isolation of
2. Smear grease (silicone) on one edge of a stainless steel, glass or poly- colonies
styrene ring.
3. Place the ring on the bottom of the petri dish, greased edge down, so
as to enclose the colony within the ring.
4. Press the ring against the bottom of the petri dish to paste the ring on
the bottom of the dish.
5. Replace the medium inside the ring with an appropriate enzyme solution (i.e., 0.5% dispase in culture medium).
6. After partial enzyme digestion, suspend the cells of the colony by gentle
pipetting into the medium in the ring.
7. Transfer the cell suspension in the ring to a centrifuge tube.
8. Centrifuge the cells at 150 g for 5 min.
1. Resuspend the cell pellet in approximately 0.5 ml of culture medium, Seeding
and transfer the cell suspension into the well of a culture plate.
2. Thereafter, follow step 3 of the maintenance procedure given in
subprotocol 1.
3. When the cell population has increased sufficiently, repeat the procedure from step 1 of "Preparation of cells" to step 2 of "Seeding".
Results
If the cells are density independent, cell colonies will be obtained even in
a low-density cell culture. Because there is possibility of contamination
with cells from other colonies, it is better to repeat the whole cloning
procedure at least once to get a reclone or re-reclone.
313
Procedure
1. Select a culture containing cells to be cloned, and harvest the cells by Preparation
treating cells with an appropriate enzyme (see chapter 4, subprotocol of cells
1-3).
2. Wash the harvested cells with culture medium by centrifugation at
150 g for 5 min.
3. Suspend the cell pellet in culture medium.
1. Pour the cloning medium into a petri dish to a depth of 3-5 mm.
Dilution of cell
2. Add the cell suspension to the petri dish so as to make a cell density of suspension
10 cells/ml or 2 cells/cm 2 •
3. Culture the cells at the same temperature as that used for culturing
the cells.
1. When the cells have multiplied and formed colonies, select a colony. Isolation of
2. Smear grease (silicone) on one edge of a stainless steel, glass or poly- colonies
styrene ring.
3. Place the ring on the bottom of the petri dish, greased edge down, so
as to enclose the colony within the ring.
4. Press the ring against the bottom of the petri dish to paste the ring on
the bottom of the dish.
5. Replace the medium inside the ring with an appropriate enzyme solution (i.e., 0.5% dispase in culture medium).
6. After partial enzyme digestion, suspend the cells of the colony by gentle
pipetting into the medium in the ring.
7. Transfer the cell suspension in the ring to a centrifuge tube.
8. Centrifuge the cells at 150 g for 5 min.
1. Resuspend the cell pellet in approximately 0.5 ml of culture medium, Seeding
and transfer the cell suspension into the well of a culture plate.
2. Thereafter, follow step 3 of the maintenance procedure given in
subprotocol 1.
3. When the cell population has increased sufficiently, repeat the procedure from step 1 of "Preparation of cells" to step 2 of "Seeding".
Results
If the cells are density independent, cell colonies will be obtained even in
a low-density cell culture. Because there is possibility of contamination
with cells from other colonies, it is better to repeat the whole cloning
procedure at least once to get a reclone or re-reclone.